Genetic Analysis of Completely Sequenced Disease-Associated MHC Haplotypes Identifies Shuffling of Segments in Recent Human History

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II–related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR–DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations

Colloid chemistry pitfall for flow cytometric enumeration of viruses in water

Flow cytomtery (FCM) has become a standard approach to enumerate viruses in water research. However, the nature of the fluorescent signal in flow cytometric analysis of water samples and the mechanism of its formation, have not been addressed for bacteriophages expected in wastewaters. Here we assess the behaviour of fluorescent DNA-staining dyes in aqueous solutions, as well as sensitivity and accuracy of FCM for enumeration of DNA-stained model bacteriophages λ, P1, and T4. We demonstrate that in aqueous systems fluorescent dyes form a self-stabilized (pseudolyophilic) emulsion of auto-fluorescing colloid particles. Sample shaking and addition of surfactants enhance auto-fluorescence due to increased dispersion and, in the presence of surfactants, stabilization of the dye emulsion. Bacteriophages with genome sizes <100 kbp (i.e. λ & P1) did not generate a distinct population signal to be detected by one of the most sensitive FCM instruments available (BD LSR Fortessa™ X-20), whereas the larger T4 bacteriophage was resolved as a distinct population of events. These results indicate that the use of fluorescent dyes for bacteriophage enumeration by flow cytometry can produce false positive signals and lead to wrong estimation of total virus counts by misreporting colloid particles as virions, depending on instrument sensitivity. Keywords: Colloid, Auto-fluorescence, FCM, Virus enumeration, SYBR® green

A lab-on-chip for malaria diagnosis and surveillance

This study examines a lab-on-chip that was created to overcome barriers in technology, reagent storage, cost and expertise in this study a simple, lab-on-chip PCR diagnostic was created for malaria testing.Background: Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges. Methods: The platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection. Results: This diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/μL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%. Conclusions: These results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries

Complete MHC Haplotype Sequencing for Common Disease Gene Mapping

The future systematic mapping of variants that confer susceptibility to common diseases requires the construction of a fully informative polymorphism map. Ideally, every base pair of the genome would be sequenced in many individuals. Here, we report 4.75 Mb of contiguous sequence for each of two common haplotypes of the major histocompatibility complex (MHC), to which susceptibility to >100 diseases has been mapped. The autoimmune disease-associated-haplotypes HLA-A3-B7-Cw7-DR15 and HLA-A1-B8-Cw7-DR3 were sequenced in their entirety through a bacterial artificial chromosome (BAC) cloning strategy using the consanguineous cell lines PGF and COX, respectively. The two sequences were annotated to encompass all described splice variants of expressed genes. We defined the complete variation content of the two haplotypes, revealing >18,000 variations between them. Average SNP densities ranged from less than one SNP per kilobase to >60. Acquisition of complete and accurate sequence data over polymorphic regions such as the MHC from large-insert cloned DNA provides a definitive resource for the construction of informative genetic maps, and avoids the limitation of chromosome regions that are refractory to PCR amplification

Microfluidic Platform for Single Nucleotide Polymorphism Genotyping of the Thiopurine S-Methyltransferase Gene to Evaluate Risk for Adverse Drug Events

Prospective clinical pharmacogenetic testing of the thiopurine S-methyltransferase gene remains to be realized despite the large body of evidence demonstrating clinical benefit for the patient and cost effectiveness for health care systems. We describe an entirely microchip-based method to genotype for common single nucleotide polymorphisms in the thiopurine S-methyltransferase gene that lead to serious adverse drug reactions for patients undergoing thiopurine therapy. Restriction fragment length polymorphism and allele-specific polymerase chain reaction have been adapted to a microfluidic chip-based polymerase chain reaction and capillary electrophoresis platform to genotype the common *2, *3A, and *3C functional alleles. In total, 80 patients being treated with thiopurines were genotyped, with 100% concordance between microchip and conventional methods. This is the first report of single nucleotide polymorphism detection using portable instrumentation and represents a significant step toward miniaturized for personalized treatment and automated point-of-care testing

LD Structure around the <i>HLA-DR</i> Region

<p>High-resolution view of the <i>HLA-DR</i> region, as represented by GOLDsurfer three-dimensional view of D′ values [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-b081" target="_blank">81</a>]. The position of the 158-kb segment shared by identical by descent between COX and QBL is shown by a dashed white line. High LD areas (red blocks) are separated by LD breaks. The first LD break (1) corresponds to a recombination hotspot mapped between <i>NOTCH4</i> and <i>C6orf10</i> in the class II–III boundary region. Another LD break (2) is visualized at another recombination hotspot centromeric of <i>HLA-DQB1</i> at the boundary of the SNP desert between COX and QBL. This is followed centromerically by a further four LD breaks corresponding to recombination hotspots mapped at <i>BRD2</i>/<i>HLA-DOA</i> interval, within <i>HLA-DMB,</i> within <i>TAP2</i> and <i>HLA-DQB2</i>/-<i>DOB</i> interval [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-b005" target="_blank">5</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-b051" target="_blank">51</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-b053" target="_blank">53</a>]. An asterisk (*) indicates a region of depleted SNP data, likely owing to substantial genotyping failure in an area with an extreme level of polymorphism.</p

Positional Distributions of Variations between COX and QBL in the <i>HLA-DR</i> Region

<div><p>MHC sequences were divided into 10-kb bins, and variations were calculated in each bin. Results are expressed as variations per 1 kb. Red and blue plots relate to SNP and DIP variations respectively.</p><p>Within a stretch of approximately 160 kb between <i>HLA-DRB3</i> and <i>HLA-DQB3,</i> only 14 SNPs and six small DIPs, comprising 1 bp, 6 bp, 10 bp (five copies of a dinucleotide repeat), and 54 bp (two copies of 27 mer), were contained. None of the variations located to coding sequence or the defined promoter regions of the HLA class II genes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-b086" target="_blank">86</a>].</p><p>Four 1-bp DIPs, labelled in grey, were identified between <i>DRB1</i> and <i>DQA1</i> where LR-PCR products were used to close a small gap resulting from clone deficit. These DIPs were located in polyA/T tracts in which the probability of <i>Taq</i> slippage in PCR products is much higher than in in-vivo amplified plasmid DNA such that their authenticity was questionable and they were excluded from analyses (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020009#pgen-0020009-sg002" target="_blank">Figure S2</a> shows one alignment of sequence traces with differing polyT tracts).</p></div

Positional Distributions of Variations between PGF and QBL and COX and QBL

<div><p>(A) Shows the distribution for PGF and QBL and (B) shows COX and QBL. MHC sequences were divided into 10-kb bins, and variations were calculated in each bin. Results are expressed as variations per 1 kb. Red and blue plots relate to SNP and DIP variations respectively. The sequence is interrupted by five gaps, shown as green vertical bars, where BACs encompassing these regions could not be identified from the clone library, which by comparison with PGF comprise a total of approximately 317 kb. The lengths and gene content of these gaps were as follows, from left to right: 159 kb including <i>OR2U1P</i> to <i>OR12D2;</i> 51 kb containing <i>HCP5;</i> 26 kb containing <i>C6orf26, C6orf27,</i> and the three exons of 3′ end of <i>MSH5;</i> 53 kb containing <i>CREBL1, FKBPL,</i> and six exons of the 5′ end of <i>TNXB;</i> and 27 kb containing <i>HLA-DOB</i>. These gaps do not represent large genomic deletions within the QBL haplotype since exonic sequence from selected genes within these regions were successfully amplified from QBL genomic DNA and sequenced to confirm their identity. The grey shaded area at the telomeric end of the map represents sequence for which overlap was not obtained and was therefore outside the area that was compared.</p><p>Boundaries of the class I, II, and III regions are shown. The positions of <i>RFP</i> and <i>KIFC1</i> that define the ends of the MHC haplotype sequencing project are indicated. Landmark genes are labelled in blue. Regions 1 and 2 are the RCCX module and the <i>HLA-DRB</i> region, respectively. The <i>HLA-DRB3</i> and <i>HLA-DQB3</i> region, which shows little variation between COX and QBL haplotypes, is shaded in orange.</p></div